Matt:LabNotes/2016-2-18

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Polyacrylamide Gel on Brain Tissue Sections with Argon Chamber Test

Purpose

  • Use argon gas chamber (sealed plastic bag) to improve polymerization of polyacrylamide gel
  • Creating a low density polyacrylamide gel surrounding the tissue can keep tissue together while allowing diffusion of DNA oligos and enzymes
  • Previously tried minimizing oxygen exposure (which inhibits polymerization) by using vacuum chamber
  • Sample dish A

Experiment

  1. Turn on UV lamp in AirClean hood for 15 minutes.
  2. Use "AB mix" from last week:
    1. 180 uL IEF 40% Acrylamide (made from powder)
    2. 20 uL Acrylamide / Bis (19:1; 38%:2%)
  3. Using a 3-cc syringe and a 0.22 micron filter, filter ~200 uL of the AB mix into a 1.5 mL microcentrifuge tube.
  4. Degas the AB mix by using vacuum and sonicator
  5. Prepare fresh 5% APS (ammonium persulfate)
    1. 5 mg APS →100 uL dH20
  6. Prepare fresh 5% TEMED (in fume hood)
    1. 2 uL TEMED → 38 uL dH20.
  7. Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C)
  8. Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels
    1. 50 uL AB mix (FILTERED)
    2. 4 uL 10% BSA
    3. 138 uL dH20
    4. 4 uL 5% TEMED
    5. 4 uL 5% APS
  9. Aspirate PBS from sample and then add 200ul gel-casting mix
  10. Put in plastic bag and seal
  11. Use vacuum to suck out air and then add argon gas
  12. Let sit in sealed bag for 30min